PROJECT SUMMARY Numerous proteins involved in cell signaling contain tandem repeats of protein-protein interaction modules belonging to the family of PDZ (PSD95-Dlg1-Zo1) domains, which recognize short amino acid motifs that are typically, but not exclusively, located at the C-terminus of a protein. The affinity and specificity of PDZ domains for canonical (C-terminal) peptide ligands has been studied extensively. While there is evidence that inter- molecular PDZ-peptide interactions can be modulated by competing intramolecular interactions, the structural basis of such autoinhibitory effects and the mechanism of activation are not well understood. To address this question, we focus on Na+/H+ exchanger regulatory factor 1 (NHERF; also known as NHERF1 or EBP50), an adapter protein involved in regulating important cell signaling events at the membrane-cytoskeleton interface of epithelial cells. NHERF contains two PDZ domains and a C-terminal ezrin-binding motif (EBM), as well as long disordered regions. The first aim of this project is to establish a linkage between the structural/dynamic properties of NHERF and its affinity for physiological interaction partners. Fluorescence-based binding assays, rapid mixing and various NMR techniques, including chemical shift analysis, paramagnetic relaxation studies and H-D exchange, will provide detailed insight into (i) the mechanism by which NHERF recognizes peptide ligands derived from the C-termini of biological interaction partners, (ii) the structural and thermodynamic basis of regulation of the binding affinity and specificity of the PDZ domains, and (iii) mechanisms of activation triggered by cytoskeletal interaction partners. The second aim is to elucidate the roles of NHERF in regulating signaling, endocytosis and degradation of the epidermal growth factor receptor (EGFR), an important oncogenic driver of many cancers. Cell-biological studies identified potential interaction sites in the C-terminal regulatory region of EGFR for the first PDZ domain of NHERF. However, there have been no biophysical studies in vitro, and the structural details and biological implications of these interactions are poorly understood. We seek to fill these gaps in knowledge (i) by using NMR and fluorescence techniques to elucidate the structural and energetic basis of interactions between NHERF and the cytoplasmic regulatory region EGFR interactions, (ii) biophysical studies of the influence of the membrane environment and effector proteins on NHERF-EGFR interactions, and (iii) by using flow cytometry and cellular imaging approaches to explore the impact of interactions with NHERF on internalization, recycling and/or degradation of EGFR in human cancer cell lines. These cell-biological approaches, combined with the structural and mechanistic insight into activation of NHERF and its interactions with EGFR gained from the proposed biophysical studies, will yield detailed new insight into the role NHERF in regulating the stability, cellular localization and cell growth-stimulating activities of EGFR.